Gestational Diabetes Mellitus (GDM) complicates up to 10% of all pregnancies and results in fetal hyperinsulinemia, macrosomia, and maternal complications during delivery. The morbidity to the mother and fetus does not end after pregnancy as recent data demonstrate a higher risk of obesity and glucose intolerance in adult offspring from GDM women and extremely high risk for type II DM in former GDM women. Our human studies have shown that a major defect in GDM involves the inability of insulin to stimulate glucose transport into skeletal muscle. The mechanisms for this severe insulin resistance are unknown, but likely involve a defect in the insulin receptor, IRS-1, and an unknown post-receptor defect at the level of GLUT4 translocation. The long-term goal of this project is to elucidate the underlying molecular signaling mechanisms that provoke insulin resistance in women with GDM. In this proposal we will dissect the roles of hormones of pregnancy, including TNFalpha, as negative regulators of insulin signaling, with a special emphasis on mechanisms for serine phosphorylation of IR and IRS-1 using human muscle fibers and L6 myotubes. Specific Aim 1 will test the hypothesis that re-distribution of PI 3-kinase to the insulin receptor mediates insulin resistance in pregnancy by triggering increased serine kinase activity to inhibit IRtyrosine phosphorylation and trigger IRS-1 degradation. In Specific Aim 2, we will determine how placental derived hormone(s) down-regulate IR and IRS-1 signaling using L6 muscle cells. In Specific Aim 3, we will investigate the contribution of skeletal muscle TNFalpha production as a mechanism for greater insulin resistance in women with GDM. In Specific Aim 4, we will explore the role of the novel CAP/Cbl signaling pathway as a potential mediator of GLUT4 translocation independent of PI 3-kinase. The outcome of these studies will provide important new insights into how pregnancy triggers insulin resistance in human skeletal muscle, and novel mechanisms for the down-regulation of insulin signaling. Ultimately, these studies should provide us with a better understanding of the cellular factors that trigger human GDM.